Immunogenic hiv-1 multi-clade, multivalent constructs and methods of their use

ABSTRACT

Described herein are nucleic acid molecules which encode multiple highly conserved epitopes from HIV-1 proteins, and optionally also epitopes from CCR5; usually also included sequences that encode spacers between two or more of the epitopes. Some of the provided nucleic acid molecules further include sequences that encode targeting domains, useful for targeting the encoded protein into a pathway for enhancing epitope presentation in a vertebrate immune system. Also described are multivalent proteins encoded for by these nucleic acid molecules. The disclosure also encompasses immunogenic compositions that comprise one or more of the nucleic acid molecules, and/or one or more of the proteins encoded thereby, as well as methods of inducing an immune response against HIV-1 in a subject by administering to the subject an effective amount of a composition containing one or more of these molecules. Also provided are cultured host cells containing within them one or more of the described nucleic acid molecules.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional PatentApplication No. 60/458,880 filed Mar. 28, 2003, which is incorporatedherein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made by the Centers for Disease Control andPrevention, an agency of the United States Government. Therefore, theU.S. Government has certain rights in this invention.

FIELD

This disclosure relates to compositions for induction of immuneresponses in vertebrates. More particularly, it relates to highlyeffective, broad spectrum multivalent constructs, both protein andnucleic acid, for inducing an immune response to an immunodeficiencyvirus, such as HIV-1. The disclosure further relates to vaccinescomprising immunogenic compounds.

BACKGROUND

Vertebrates have developed a sophisticated system to protect themselvesagainst a wide variety of hazards including various viruses andmicroorganisms, such as bacteria and fungi, as well as genetic diseases,neoplasia, and effects of a variety of toxins. The system has evolvedbased on the ability to recognize self as distinct from non-self or“foreign.” A broad panoply of defense mechanisms are involved, includingphagocytosis, lysis, such as complement mediated or perform mediatedlysis, and killer cells, such as cytotoxic T-lymphocytes (CTLs; alsoknown as cytotoxic/suppressor T-cells, Tc/s), natural killer cells,antibody dependent cytotoxic cells, and the like. Various cell typesoffer different mechanisms whereby the invader or endogenous diseasedcell may be eliminated.

A key to the immune defensive mechanism is the T-cell. For instance, itis well known that the adaptive immune system shows a much strongerresponse on second, as compared to first, encounter with an antigen.This phenomenon is exploited in vaccination, which works by inducing astate of lasting immunity known as immunological memory. Immunologicalmemory requires activation of T-lymphocytes specific for the vaccineantigen.

T-cells have been found to be “restricted” in that they respond to anantigen in relation to one or a few specific molecules (now called majorhistocompatibility or MHC molecules) associated with their natural host.In vitro, T-cells from a host of one haplotype respond to an antigen inrelation to an MHC molecule of a different haplotype host. The T-cellreceptor recognition repertoire appears to be narrower than therecognition repertoire of immunoglobulins produced by B-cells. Inaddition, rather than directly binding to an antigen as do antibodiesand other immunoglobulins, the T-cell receptor appears to requireconcomitant binding to a foreign antigen and an MHC molecule.

MHC molecules are divided into two classes, Class I and Class II. Theformer class is relatively ubiquitous on vertebrate cells, while thelatter is generally limited to lymphocytes, macrophages, and dendriticcells. Functionally different T-cells appear to be activated in relationto one or the other class of MHC molecules. The nature of the activityof a T-cell varies with the Class of the MHC molecule to which it iscomplementary. A T-cell clone recognizes a specific antigen inconjunction with a specific MHC allele. Furthermore, variation in theantigen structure affects the nature of the response when the T-cell,antigen, and antigen presenting cell are brought together. Dependingupon the nature of the structural change, three possibilities areencountered: no change, increased stimulation or decreased stimulationof an immune response to the antigen.

T-lymphocytes detect foreign polypeptide antigens by recognizing—via theT-cell receptor (“TCR”)—peptide fragments derived from the antigen. MostT-lymphocytes, however, are MHC restricted, that is, they recognize onlycomplexes of peptides bound to the highly polymorphic membrane proteinsencoded by Class I and Class II MHC genes and presented (displayed) onthe surface of an accessory cell (designated an antigen-presenting cellor “APC”), in which the antigen has been processed.

Antigens can be processed by one of two pathways, depending on theirorigin, inside or outside the APC. In a first pathway, foreign materialfrom outside a cell is engulfed by a specialized APC (often a macrophageor B-cell), which breaks down the material and complexes the processedantigen with Class II MHC molecules. In particular, MHC Class IImolecules are synthesized in the endoplasmic reticulum with theirantigenic peptide binding sites blocked by the invariant chain protein(Ii). These MHC Class II-Ii protein complexes are transported from theendoplasmic reticulum to a post-Golgi compartment where Ii is releasedby proteolysis and a specific antigenic peptide becomes bound to the MHCClass II molecule.

Class II MHC molecules are expressed primarily on cells involved ininitiating and sustaining immune responses, such as T-lymphocytes,B-lymphocytes, and macrophages. Complexes of Class II MHC molecules andimmunogenic peptides are recognized by helper T-lymphocytes (also knownas helper/accessory T-cells, “Th”) and induce proliferation of Thlymphocytes. Class II MHC complexes also stimulate secretion ofcytokines by Th cells, resulting in amplification of the immune responseto the particular immunogenic peptide that is displayed. Th1 cellsproduce interferon-γ and other cytokines that stimulate CTLs, whileother cytokines produced by Th2 cells help B-cells to produceantibodies.

A second antigen processing pathway is generally involved with foreignor aberrant proteins made within cells, such as virus-infected ormalignant cells. These proteins are subjected to partial proteolysis bythe proteosome within such cells, so as to form peptide fragments thatthen associate with Class I MHC molecules and are transported to thecell surface for presentation to T-cells. Class I MHC molecules areexpressed on almost all nucleated cells, and complexes of Class I MHCmolecules and bound immunogenic peptides are recognized by CTLs, whichthen destroy the antigen-bearing cells. CTLs are particularly importantin tumor rejection and in fighting viral infections.

For a CTL to recognize an antigen in the form of a peptide fragmentbound to the MHC class I molecule, that antigen must normally beendogenously synthesized by the cell and a portion degraded into smallpeptide fragments in the cytoplasm. Some of these small peptidestranslocate into a pre-Golgi compartment and interact with Class I heavychains to facilitate proper folding and association with the subunit 132microglobulin. The peptide-MHC Class I complex is then routed to thecell surface for expression and potential recognition by specific CTLs.

By these dual antigen processing pathways, appropriate defenses aregenerated against both exogenous and internally produced antigens. Thus,antigens taken up from the extracellular environment eventually elicitB-cells to produce antibodies that protect the organism against asubsequent challenge by an agent comprising the exogenous antigen. Onthe other hand, antigens comprised of abnormal structures made within anabnormal or errant cell (for example a virus-infected or malignant cell)activate an immune response that eventually leads to killing of theerrant cell. There is considerable interest in methods for betterstimulating immune responses to antigens that are processed by either ofthese two pathways and presented by either MHC Class I or Class IImolecules.

In view of the above knowledge, it is understandable that there has beensubstantial interest in using short peptides to affect an immuneresponse in vivo and in vitro, to provide stimulation or inactivation ofa particular response. Thus, appropriate immunogenic peptides mightmodulate a natural immune response to a particular event, either byactivating particular lymphocytes to enhance a protective response or bydeactivating particular lymphocytes to diminish or prevent anundesirable response.

The human immunodeficiency virus (HIV-1, also referred to as HTLV-III,LAV or HTLV-III/LAV) is the etiological agent of the acquired immunedeficiency syndrome (AIDS) and related disorders (see, for example,Barre-Sinoussi et al., Science 220:868-871, 1983; Gallo et al., Science224:500-503, 1984; Levy et al., Science 225:840-842, 1984; Siegal etal., N. Engl. J. Med 305:1439-1444, 1981). AIDS patients usually have along asymptomatic period followed by the progressive degeneration of theimmune system and the central nervous system. Replication of the virusis highly regulated, and both latent and lytic infection of the CD4positive helper subset of T-lymphocytes occur in tissue culture (Zaguryet al., Science 231:850-853, 1986). Molecular studies of HIV-1 show thatit encodes a number of genes (Ratner et al., Nature 313:277-284, 1985;Sanchez-Pescador et al., Science 227:484-492, 1985), including threestructural genes—gag, pol and env—that are common to all retroviruses.Nucleotide sequences from viral genomes of other retroviruses,particularly HIV-2 and simian immunodeficiency viruses (SIV; previouslyreferred to as STLV-III), also contain these structural genes (Guyaderet al., Nature 326:662-669, 1987; Chakrabarti et al., Nature328:543-547, 1987).

Development of an effective HIV vaccine is a major challenge due toantigenic variation and immune escape mechanisms. Strategies thatinclude the use of recombinant DNA technology and novel antigen deliverymethods are being applied to the development of HIV vaccines. Most HIV-1vaccine constructs (DNA and recombinant protein vaccine) aresubtype-specific and designed to prime only one arm of the immunesystem, that is, CTL responses or humoral B-cell responses. Emergingdata suggest that broadly reactive T-cell responses, as well asneutralizing antibody responses are likely to be required for aneffective immune response against HIV-1. Additionally, current humanphase III vaccine trials using recombinant envelope proteins, suggestthat immunity to HIV-1 envelope proteins is probably not sufficient forcomplete protection against HIV-1. Thus the results from multiplestudies suggest that additional epitopes as well as activation of botharms of the immune system may be required for an effective HIV-1vaccine.

By way of one example of peptide immunogens, Peter et al. (Vaccine19:4121-4129, 2001) disclose induction of a CTL response againstmultiple CTL epitopes present in HIV proteins using short syntheticpeptides. Four IHLA-A2.1 restricted peptides (RT 476-484, p17 77-85,gp41 814-823, RT 956-964) that showed stable binding to the HLA-A2.1molecule in an in vitro binding assay were able to elicit a strongspecific immune response in HLA-A2.1 transgenic mice when injected witha peptide (“P30”) used as a universal T-cell helper epitope, inincomplete Freund adjuvant (IFA) or a nonionic emulsifier (Montanide™ISA 720). The use of biodegradable poly-L-glutamic acid (PLGA)microspheres (MS) as adjuvant was also successfully tested for allpeptides.

Many studies of cross-clade recognition of HIV epitopes have beencarried out (see, for example, Wilson et al., AIDS Res. Hum.Retroviruses 14:925-937, 1998; McAdam et al., AIDS 12:571-579, 1998;Lynch et al., J Infect Dis. 178:1040-1046, 1998; Boyer et al. Dev. Biol.Stand. 95:147-53, 1998; Cao et al., J. Virol. 71:8615-8623, 1997; Duraliet al., Viral. 72:3547 3553, 1998). These studies often used whole-gene,vaccinia-expressed constructs to probe CTL lines from HIV-1 infected orHIV-1 vaccinated volunteers for CTL responses. What appeared to becross-clade recognition by CTLs in these experiments may have beenrecognition of CTL epitopes that are conserved within the large geneconstructs cloned into the vaccinia constructs and into the vaccinestrain (or the autologous strain). Where responses to specific peptides,and their altered sequences in other HIV strains, have been tested, andthe peptides have been mapped, some studies have shown a lack ofcross-strain recognition (Dorrel et al., HIV Vaccine DevelopmentOpportunities And Challenges Meeting, Abstract 109 (Keystone, Colo.,January 1999)). Studies of virus escape from CTL recognition carried outon HIV-1 infected individuals have also shown that viral variation atthe amino acid level may abrogate effective CTL responses (Koup, J. Exp.Med. 180:779-782, 1994; Dai et al., J. Virol. 66:3151-3154, 1992;Johnson et al., J. Exp. Med. 175:961-971, 1992).

Unfortunately, existing candidate HIV-1 vaccines are subtype specific,and are expected not protect against diverse natural HV-1 infections.This is true of both DNA vaccine constructs as well as recombinantprotein vaccines. Furthermore, many of the existing constructs havefocused on priming only one arm of the immune system, that is, cellmediated T-cell responses or humoral B-cell responses. In addition,while some DNA constructs have shown promising results in loweringviremia in animal model systems, none has been able to confersterilizing immunity. These data suggest that both B-cell and T-cellresponses may be needed for a protective immune response against HIV-1.Additionally, current human phase 3 vaccine trials using recombinantenvelope proteins, suggests that immunity to HIV-1 envelope proteins isprobably not sufficient for complete protection against HIV-1.Prime-boost strategy using recombinant envelope from HIV-1 subtype Balso has not been successful in boosting the immune responses.

As the HIV epidemic continues to spread world wide, the need foreffective immune-stimulatory compositions and vaccines remains urgent.

SUMMARY OF THE DISCLOSURE

Multi-clade multivalent (MCMV) (polyepitope; multi-epitope) polypeptidesand mixtures of polypeptides have been developed, which can be used tostimulate immune responses to HIV-1 in vertebrates. In variousembodiments, these polypeptides and polypeptide mixtures includeimmunogenic CTL, T- and/or B-cell determinants that are capable ofeliciting broad and effective immune responses against diverse subtypesof HIV-1. Immunogens described herein are designed to besubtype-independent and will provide both prime and boost reagents forworldwide use.

Also described herein are recombinant MCMV constructs that can be useddirectly or indirectly to protect subjects against infection by multipleHIV-1 subtypes. These constructs are designed to elicit T-cell, B-cell,or both T-cell and B-cell responses against highly conserved epitopeswithin multiple HIV-1 subtypes. The constructs, when integrated into avector, can be used as immunogens, can be used as DNA vaccines, and canbe used as sources of recombinant protein for stimulation of immuneresponses in subjects, as well as for protein boosts to subjects whohave received a nucleic acid construct previously.

Without being bound by theory, it is believed that the MCMV HIV-1constructs and polypeptides provide universal immune stimulants andvaccines, capable of effective use in any part of the world affected bythe HIV-1 epidemic.

The construction and design of specific provided constructs areparticularly useful in that they allow convenient addition/deletion ofepitopes, and contain specific cellular targeting domains that optimizeantigen processing and recognition.

The provided constructs and proteins encoded thereby also can becombined with other epitope-based constructs to generate, for instance,multi-pathogen vaccines.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a series of schematic drawings of embodiments of specificmulti-clade, multivalent gene constructs.

FIG. 1A shows one embodiment of a CTL-stimulating MCMV (MCMV-CTL)construct. Epitopes were chosen based on prior responses of HIV+individuals, predicted HLA binding and sequence conservation amongmultiple HIV subtypes. In the Examples provided below, such a geneconstruct was assembled using synthetic single stranded oligonucleotides(100-130 mers) that contain strings of 3-6 CTL epitopes and linker aminoacid sequences (exemplified by the tri-amino acid KAA), which wereincluded to improve processing of epitopes. A modified human ubiquitinpeptide is optionally added to the amino terminus of the molecule tofurther increase CTL epitope processing.

FIG. 1B shows one embodiment of a MCMV-AB/Th construct. Antibody andT-helper epitopes conserved among multiple subtypes of HIV-1 were chosenand single stranded oligos (100-120 mers) for these epitopes weresynthesized. The lysosomal integral membrane protein-II (LIMP-II) signalsequence is optionally included to enhance processing of T-helperepitopes.

FIG. 1C is a schematic illustration of a MCMV-CTL, illustrating that thesame nucleic acid construct can be used to generate both ubiquitin+ andubiquitin-sequences by differential placement of the forward primer usedto amplify the sequence.

FIG. 1D shows an alternative embodiment, in which both the CTL and theAB/Th epitopes are provided in the same recombinant construct. In acombined construct such as this, the order of the different epitope setscan be rearranged.

FIG. 2 is a schematic illustration of the assembly of representativeMCMV construct. In the illustrated embodiment (FIG. 2A), overlappingsingle stranded oligonucleotides (100-130 mers), spanning the fulllength of the MCMV-CTL-ubiquitin construct (1.5 kb) were synthesized(eight forward and eight reverse). Through a series of splicing overlapextension (SOE), polymerase chain reaction (PCR) and cloning steps, a1,553 base pair recombinant nucleic acid sequence was generated and thencloned into pVax-1 (Invitrogen, Carlsbad, Calif.) (FIG. 2B).Alternatively, the construct was also assembled without includingubiquitin (FIG. 2C).

FIG. 3 is an schematic drawing of vector PTriex-4 (Novagen, Madison,Wis.), which contains a representative MCMV-CTL construct, which can beused for production of recombinant protein in either bacterial,mammalian or insect cells.

FIG. 4 is a western blot analysis showing expression of aMCMV-CTL-ubiquitin polypeptide fusion construct in E. coli. The fusionprotein is predicted to be 64 kDa (57 kDa plus the 7 kDa expressiontag); expressed protein is indicated in the right hand most lane.

FIG. 5 is a western blot analysis of extract from HeLa cells transfectedwith pVax-1 (Invitrogen, Carlsbad, Calif.) containing theMCMV-CTL-ubiquitin sequence, using an anti-ubiquitin antibody fordetection. Cells were transfected using various concentrations ofGeneJuice reagent (Merck Biosciences, San Diego, Calif.) with 1 μg ofDNA. Cells were harvested 24 and 48 hours post transfection. Aubiquitinated protein of the correct predicted molecular weight (˜56kDa) of the synthetic MCMV-CTL-ubiquitin construct is clearly visible,as is normal cellular ubiquitin (˜10 kDa).

FIG. 6 is a series of bar graphs demonstrating breadth and magnitude ofCTL responses observed to peptides contained in the MCMV-CTL constructwith peripheral blood mononuclear cells (PBMCs) from individualschronically infected with HIV-1 subtype B. The responses are reported asspot forming cells per 105 PBMCs.

FIG. 7 is a bar graph demonstrating percentage predicted epitoperecognition based on subject HLA type. The percentage of predictedepitopes that were targeted by patients' CD8+cells in the Elispot assayis shown.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO: 1 shows the nucleic acid sequence and amino acid sequence ofMCMV-CTL-ubiquitin. The nucleic acid sequence includes uniquerestriction sites at positions 6-11 and 1548-1553. These restrictionsites can be used to insert the epitope construct into differentvectors.

SEQ ID NO: 2 shows the amino acid sequence of MCMV-CTL-ubiquitin.Ubiquitin is positions 1-76. The “KAA” spacer peptide appears (aminoacid positions 117-119, 169-172, 242-244, 288-290, 317-319, 367-369,417-419, and 466-468) throughout the remainder of the sequence betweenstrings of three to five CTL epitopes.

SEQ ID NO: 3 shows the nucleic acid sequence and amino acid sequence ofMCMV-CTL (no ubiquitin). The nucleic acid sequence includes uniquerestriction sites at positions 1-6 and 1318-1323. These restrictionsites can be used to insert the epitope construct into differentvectors.

SEQ ID NO: 4 shows the amino acid sequence of MCMV-CTL (no ubiquitin).The “KAA” spacer appears throughout the sequence, at positions analogousto those in SEQ ID NO: 2.

SEQ ID NO: 5 shows the amino acid sequence of CTLUbiquitinNC (withoutmouse and monkey control epitopes).

SEQ ID NO: 6 shows the amino acid sequence of CTLNC (no ubiquitin,without mouse and monkey control epitopes).

SEQ ID NO: 7 shows the nucleic acid sequence and amino acid sequence ofMCMV-AB/Th with LIMP-II.

SEQ ID NO: 8 shows the amino acid sequence of MCMV-AB/Th with LIMP-II.

SEQ ID NO: 9 shows the nucleic acid sequence and amino acid sequence ofMCMV-AB/Th without LIMP-II.

SEQ ID NO: 10 shows the amino acid sequence of MCMV-AB/Th withoutLIMP-II.

SEQ ID NOs: 11-22 show the amino acid sequences of additional HIV-1 CTLantigenic fragments/epitopes.

SEQ ID NOs: 23-45 show the amino acid sequences of control peptides.

SEQ ID NOs: 46-59 show the amino acid sequences of additional HIV-1B-cell antigenic fragments/epitopes.

SEQ ID NOs: 60-64 show the amino acid sequences of additional HIV-1T-helper cell antigenic fragments/epitopes.

DETAILED DESCRIPTION

I. Abbreviations HIV human immunodeficiency virus LIMP-II lysosomalintegral membrane protein II MCMV multi-clade multivalent MCMV-AB/ThB-cell/T-cell epitopes MCMV construct/polypeptide MCMV-CTL CTL epitopesMCMV construct/polypeptide PCR polymerase chain reaction SOE splicingoverlap extensionII. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

Adjuvant: A substance that non-specifically enhances the immune responseto an antigen. Development of vaccine adjuvants for use in humans isreviewed in Singh et al., Nat. Biotechnol. 17:1075-1081, 1999, whichdiscloses that, at the time of its publication, aluminum salts and theMF59 microemulsion are the only vaccine adjuvants approved for humanuse.

Binding or stable binding (of an oligonucleotide): An oligonucleotidebinds or stably binds to a target nucleic acid if a sufficient amount ofthe oligonucleotide forms base pairs or is hybridized to its targetnucleic acid, to permit detection of that binding. Binding can bedetected by either physical or functional properties of thetarget:oligonucleotide complex. Binding between a target and anoligonucleotide can be detected by any procedure known to one skilled inthe art, including both functional and physical binding assays. Bindingmay be detected functionally by determining whether binding has anobservable effect upon a biosynthetic process such as expression of agene, DNA replication, transcription, translation and the like.

Physical methods of detecting the binding of complementary strands ofDNA or RNA are well known in the art, and include such methods as DNaseI or chemical footprinting, gel shift and affinity cleavage assays,Northern blotting, dot blotting and light absorption detectionprocedures. For example, one method that is widely used, because it isso simple and reliable, involves observing a change in light absorptionof a solution containing an oligonucleotide (or an analog) and a targetnucleic acid at 220 to 300 nm as the temperature is slowly increased. Ifthe oligonucleotide or analog has bound to its target, there is a suddenincrease in absorption at a characteristic temperature as theoligonucleotide (or analog) and target disassociate from each other, ormelt.

The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of theoligomer is melted from its target. A higher (T_(m)) means a stronger ormore stable complex relative to a complex with a lower (T_(m)).

Complementarity and percentage complementarity: Molecules withcomplementary nucleic acids form a stable duplex or triplex when thestrands bind, (hybridize), to each other by forming Watson-Crick,Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when anoligonucleotide remains detectably bound to a target nucleic acidsequence under the required conditions.

Complementarity is the degree to which bases in one nucleic acid strandbase pair with the bases in a second nucleic acid strand.Complementarity is conveniently described by percentage, that is, theproportion of nucleotides that form base pairs between two strands orwithin a specific region or domain of two strands. For example, if 10nucleotides of a 15-nucleotide oligonucleotide form base pairs with atargeted region of a DNA molecule, that oligonucleotide is said to have66.67% complementarity to the region of DNA targeted.

A thorough treatment of the qualitative and quantitative considerationsinvolved in establishing binding conditions that allow one skilled inthe art to design appropriate oligonucleotides for use under the desiredconditions is provided by Beltz et al., Methods Enzymol. 100:266-285,1983, and by Sambrook et al. (ed), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide, orfor a stop signal. The term codon is also used for the corresponding(and complementary) sequences of three nucleotides in the mRNA intowhich the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule. Thus, a reference to the nucleic acidmolecule that encodes a particular MCMV construct, or a fragmentthereof, encompasses both the sense strand and its reverse complement.Thus, for instance, it is appropriate to generate probes or primers fromthe reverse complement sequence of the disclosed nucleic acid molecules.

Deletion: The removal of a sequence of DNA, the regions on either sideof the removed sequence being joined together. Similar, this term canrefer to the removal (for example, though genetic engineering means) ofan amino acid sequence within a protein, the regions on either side ofthe removed sequence being joined together.

Epitope tags: Short stretches of amino acids to which a specificantibody can be raised, which in some embodiments allows one tospecifically identify and track the tagged protein that has been added(for instance) to a living organism or to cultured cells. Detection ofthe tagged molecule can be achieved using a number of well knowntechniques. Examples of such techniques include (but are not limitedto): immunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, ELISA, immunoblotting (“western”blotting), and affinity chromatography. Examples of well known epitopetags include FLAG, T7, HA (hemagglutinin) and myc. The FLAG tag(DYKDDDDK) is beneficially used in some embodiments because high qualityreagents are available to be used for its detection.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidconsists of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas “base pairing.” More specifically, A will hydrogen bond to T or U,and G will bond to C. “Complementary” refers to the base pairing thatoccurs between to distinct nucleic acid sequences or two distinctregions of the same nucleic acid sequence.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide (or its analog)and the DNA or RNA target. The oligonucleotide or oligonucleotide analogneed not be 100% complementary to its target sequence to be specificallyhybridizable. An oligonucleotide or analog is specifically hybridizablewhen binding of the oligonucleotide or analog to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide or analog to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ concentration) of the hybridization bufferwill determine the stringency of hybridization, though wash times alsoinfluence stringency. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, chapters 9 and 11, herein incorporated by reference.

For purposes of the present disclosure, “stringent conditions” encompassconditions under which hybridization will only occur if there is lessthan 25% mismatch between the hybridization molecule and the targetsequence. “Stringent conditions” may be broken down into particularlevels of stringency for more precise definition. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 25% sequence mismatch will not hybridize; conditions of“medium stringency” are those under which molecules with more than 15%mismatch will not hybridize, and conditions of “high stringency” arethose under which sequences with more than 10% mismatch will nothybridize. Conditions of “very high stringency” are those under whichsequences with more than 6% mismatch will not hybridize.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, that is, otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Lysosomal compartment: Membrane-bound acidic vacuoles containinglysosomal-associated membrane protein (LAMP) molecules in the membrane,hydrolytic enzymes that function in antigen processing, and MHC class IImolecules for antigen recognition and presentation. This compartmentfunctions as a site for degradation of foreign materials internalizedfrom the cell surface by any of a variety of mechanisms includingendocytosis, phagocytosis and pinocytosis, and of intracellular materialdelivered to this compartment by specialized autolytic phenomena (deDuve, Eur. J. Biochem. 137:391, 1983).

The biosynthesis and vacuolar targeting mechanisms of the hydrolyticenzymes present in the lysosomal compartment have been extensivelystudied (Kornfeld & Mellman, Ann. Rev. Cell Biol., 5:483, 1989). Newlysynthesized hydrolases in the Golgi apparatus acquire mannose6-phosphate groups that serve as specific recognition markers for thebinding of these enzymes to mannose 6-phosphate receptors which are thentargeted in some unknown manner to a prelysosomal vacuole. There thereceptor-enzyme complex is dissociated by low pH, and the receptorsrecycle to the Golgi apparatus, while the enzyme-containing vacuolematures into a lysosome.

Studies of the structure and function of the lysosomal membrane wereinitiated in 1981 by August and colleagues with the discovery of majorcellular glycoproteins that were subsequently termed LAMP-1 and LAMP-2due to their predominant localization in the lysosomal membrane.Analogous proteins were subsequently identified in rat, chicken andhuman cells. Typically, LAMP-1, as deduced from a cDNA clone (Chen etal., J. Biol. Chem., 263:8754, 1988) consists of a polypeptide core ofabout 382 amino acids (˜42,000 Da) with a large (346-residue)intraluminal amino-terminal domain followed by a 24-residue hydrophobictransmembrane region and short (12-residue) carboxyl-terminalcytoplasmic tail. The intraluminal domain is highly glycosylated, beingsubstituted with about 20 asparagine linked complex-typeoligosaccharides and consists of two ˜160-residue homology units thatare separated by a proline/serine-rich region. Each of these homologousdomains contains four uniformly spaced cysteine residues, disulfidebonded to form four 36-38-residue loops symmetrically placed within thetwo halves of the intraluminal domain (Arterburn et al., J. Biol. Chem.,265:7419, 1990). The LAMP-2 molecule is highly similar to LAMP-1 inoverall amino acid sequence (Cha et al., J. Biol. Chem., 265:5008,1990).

Another glycoprotein, described as CD63, MEA491 or LIMP-I, is also foundin lysosomal membranes, as well as other in vacuolar structures (Azorzaet al., Blood, 78:280, 1991). This molecule is distinctly different fromthe LAMPs, with a core polypeptide of about 25,000 Da and fourtransmembrane domains, but it has a cytoplasmic structure and sequencesimilar to the LAMP molecules. There is also extensive amino acidsequence similarity between this protein and a family of other moleculesthat also contain four membrane spanning domains, including theSchistosoma mansoni membrane protein SM23, CD37, the tumor-associatedantigen CO-029, and the target of antiproliferative antibody-1.

LIMP-II is an additional glycoprotein present in the membrane oflysosomes and secretory granules with lysosomal properties (Vega et al.,J. Biol. Chem., 266:16818, 1991). A sequence near the amino-terminuswith properties of an uncleavable signal peptide and a hydrophobic amineacid segment near the carboxyl end suggest that the protein is anchoredin cell membranes at two sites by two short cytoplasmic tails at theamine and carboxyl-terminal ends of the protein. The molecule does nothave sequence homology to any of the other described lysosomal membraneprotein, but is highly similar to the cell surface protein CD36, whichis involved in cell adhesion.

Nucleotide: This term includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by native phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain non-naturallyoccurring portions, such as altered sugar moieties or inter-sugarlinkages, such as a phosphorothioate oligodeoxynucleotide. Functionalanalogs of naturally occurring polynucleotides can bind to RNA or DNA,and include PNA molecules.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 200 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 bases, for example atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long,or from about 6 to about 50 bases, for example about 10-25 bases, suchas 12, 15 or 20 bases.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Open reading frame: A series of nucleotide triplets (codons) coding foramino acids without any internal termination codons. These sequences areusually translatable into a peptide/polypeptide/protein.

Parenteral: Administered outside of the intestine, for example, not viathe alimentary tract. Generally, parenteral formulations are those thatwill be administered through any possible mode except ingestion. Thisterm especially refers to injections, whether administeredintravenously, intrathecally, intramuscularly, intraperitoneally, orsubcutaneously, and various surface applications including intranasal,intradermal, and topical application, for instance.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful with compositions described herein are conventional.Martin, Remington's Pharmaceutical Sciences, published by MackPublishing Co., Easton, Pa., 19th Edition, 1995, describes compositionsand formulations suitable for pharmaceutical delivery of the nucleotidesand proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or other reporter molecule. Typical labels includeradioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, and enzymes. Methodsfor labeling and guidance in the choice of labels appropriate forvarious purposes are discussed, for example, in Sambrook et al. (ed.),Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 andAusubel et al. Short Protocols in Molecular Biology, 4^(th) ed., JohnWiley & Sons, Inc., 1999.

Primers are short nucleic acid molecules, for instance DNAoligonucleotides 10 nucleotides or more in length, for example thathybridize to contiguous complementary nucleotides or a sequence to beamplified. Longer DNA oligonucleotides may be about 15, 20, 25, 30 or 50nucleotides or more in length. Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then the primerextended along the target DNA strand by a DNA polymerase enzyme. Primerpairs can be used for amplification of a nucleic acid sequence, forexample, by the PCR or other nucleic-acid amplification methods known inthe art. Other examples of amplification include strand displacementamplification, as disclosed in U.S. Pat. No. 5,744,311;transcription-free isothermal amplification, as disclosed in U.S. Pat.No. 6,033,881; repair chain reaction amplification, as disclosed in WO90/01069; ligase chain reaction amplification, as disclosed in EP-A-320308; gap filling ligase chain reaction amplification, as disclosed inU.S. Pat. No. 5,427,930; and NASBA™ RNA transcription-freeamplification, as disclosed in U.S. Pat. No. 6,025,134.

Methods for preparing and using nucleic acid probes and primers aredescribed, for example, in Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989; Ausubel et al. Short Protocols inMolecular Biology, 4^(th) ed., John Wiley & Sons, Inc., 1999; and Inniset al. PCR Protocols, A Guide to Methods and Applications, AcademicPress, Inc., San Diego, Calif., 1990. Amplification primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, ©1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). One of ordinaryskill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, in order to obtaingreater specificity, probes and primers can be selected that comprise atleast 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of atarget nucleotide sequences.

Protein: A biological molecule, particularly a polypeptide, expressed bya gene and comprised of amino acids.

Purified: The term “purified” does not require absolute purity (forexample, the absence of all other substances); rather, it is intended asa relative term. Thus, for example, a purified protein preparation isone in which the protein referred to is more pure than the protein inits natural environment within a cell or within a production reactionchamber (as appropriate).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, for example, by genetic engineering techniques.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a protein-specific binding agent bindssubstantially only the defined protein, or to a specific region withinthe protein. As used herein, a protein-specific binding agent includesantibodies and other agents that bind substantially to a specifiedpolypeptide. The antibodies may be monoclonal or polyclonal antibodiesthat are specific for the polypeptide, as well as immunologicallyeffective portions (“fragments”) thereof.

Antibodies may be produced using standard procedures described in anumber of texts, including Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999. The determination that aparticular agent binds substantially only to the target protein mayreadily be made by using or adapting routine procedures. One suitable invitro assay makes use of the Western blotting procedure (described inmany standard texts, including Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999). Western blotting may be usedto determine that a given target protein binding agent, such as amonoclonal antibody, binds substantially only to the specified targetprotein.

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bindto target protein (or epitope within a protein or fusion protein) wouldalso be specific binding agents for that protein (or epitope). Theseantibody fragments are defined as follows: (1) FAb, the fragment whichcontains a monovalent antigen-binding fragment of an antibody moleculeproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) FAb′, thefragment of an antibody molecule obtained by treating whole antibodywith pepsin, followed by reduction, to yield an intact light chain and aportion of the heavy chain; two FAb′ fragments are obtained per antibodymolecule; (3) (FAb′)₂, the fragment of the antibody obtained by treatingwhole antibody with the enzyme pepsin without subsequent reduction; (4)F(Ab′)₂, a dimer of two FAb′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. Methods of making these fragments are routine.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun or other acceleration techniques (for example, air gun).

Vaccine: A term used herein to mean a composition useful for stimulatinga specific immune response (or immunogenic response) in a vertebrate. Insome embodiments, the immunogenic response is protective or providesprotective immunity, in that it enables the vertebrate animal to betterresist infection with or disease progression from the organism againstwhich the vaccine is directed. Without wishing to be bound by theory, itis believed that an immunogenic response may arise from the generationof neutralizing antibodies, T-helper, or cytotoxic cells of the immunesystem, or all of the above.

In some embodiments, an “effective amount” or “immune-stimulatoryamount” of a vaccine or vaccinating composition is an amount which, whenadministered to a subject, is sufficient to engender a detectable immuneresponse. Such a response may comprise, for instance, generation of anantibody specific to one or more of the epitopes provided in thevaccine. Alternatively, the response may comprise a T-helper orCTL-based response to one or more of the epitopes provided in thevaccine. All three of these responses may originate from naïve or memorycells. In other embodiments, a “protective effective amount” of avaccine or vaccinating composition is an amount which, when administeredto a subject, is sufficient to confer protective immunity upon thesubject.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Virus: Microscopic infectious organism that reproduces inside livingcells. A virus typically consists essentially of a core of a singlenucleic acid surrounded by a protein coat, and has the ability toreplicate only inside a living cell. “Viral replication” is theproduction of additional virus by the occurrence of at least one virallife cycle. A virus may subvert the host cells' normal functions,causing the cell to behave in a manner determined by the virus. Forexample, a viral infection may result in a cell producing a cytokine, orresponding to a cytokine, when the uninfected cell does not normally doso.

“Retroviruses” are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a retrovirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. The integrated DNAintermediate is referred to as a provirus. The term “lentivirus” is usedin its conventional sense to describe a genus of viruses containingreverse transcriptase. The lentiviruses include the “immunodeficiencyviruses” which include human immunodeficiency virus type 1 and type 2,simian immunodeficiency virus, and feline immunodeficiency virus.

HIV is a retrovirus that causes immunosuppression in humans (HIVdisease), and leads to a disease complex known as the acquiredimmunodeficiency syndrome. “HIV disease” refers to a well-recognizedconstellation of signs and symptoms (including the development ofopportunistic infections) in persons who are infected by an HIV virus,as determined by antibody detection using ELISA or western blot studies.Alternatively, HIV infection can be detected by the presence of HIV RNA(for example, using RT-PCR) or HIV integrated DNA (for example, usingPCR). Laboratory findings associated with this disease are a progressivedecline in T-helper cells and a rise in viremia (viral load asdetermined by, for instance, RT-PCR).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a”, “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. For example, the term “acell” includes a plurality of cells, including mixtures of two or moretypes of cells. The term “a nucleic acid molecule” includes a pluralityof nucleic acid molecules, or a mixture of different nucleic acidmolecules. Similarly, the same holds for “a protein” or “a polypeptide.”

As used herein, the term “comprising” shall mean that the compositionsand methods include the recited elements, but do not exclude otherelements. “Consisting essentially of” shall mean excluding otherelements of any essential significance to the combination. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and/or pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and/or excludingsubstantial additional method steps. Embodiments defined by each ofthese transition terms are within the scope.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides, are approximate and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Overview of Several Embodiments

Provided herein in various embodiments are multi-clade, multivalentrecombinant polyepitope polypeptides, which are useful to induceimmunogenic responses in vertebrate animals to HIV-1. These polypeptidesinclude CTL-stimulatory epitopes, T-helper cell stimulatory epitopes,B-cell stimulatory epitopes, or combinations of two or more such typesof epitopes. Epitopes in the polyepitope polypeptides are selected toprovide multi-clade coverage. In particular, epitopes are selected to beat least 50% conserved across a plurality of HIV-1 subtypes, forinstance, at least 2, 3, 4, 5, 6, or more HIV-1 subtypes. In particularembodiments, at least 30% of the epitopes included in a singlepolyepitope polypeptide are at least 60% conserved, at least 70%conserved, at least 80% conserved, or even more highly conserved acrossa plurality of HIV-1 subtypes.

In specific embodiments there are provided isolated polyepitopepolypeptides, wherein adjacent polypeptide segments are linked by aspacer peptide. In some examples, the spacer peptide links multiplegroups of polypeptide segments. Specific, non-limiting examples of thespacer peptide include the tri-amino acid lysine-alanine-alanine, orproline-glycine-proline.

In other examples, the isolated polyepitope polypeptides also include atargeting signal that targets the polyepitope polypeptides to a lysosomeor to a proteosome. Specific, non-limiting, examples of the targetingsignal include a targeting-competent fragment of lysosomal integralmembrane protein-II or ubiquitin.

In still other examples, the isolated polyepitope polypeptides alsoinclude a plurality of amino acid segments from one or more HIV-1coreceptors. A specific, non-limiting, example of a HIV-1 coreceptor isCCR5.

In further examples, the isolated polyepitope polypeptides include humancytotoxic T-lymphocyte stimulatory epitopes, human T-helper cellstimulatory epitopes, human B-cell stimulatory epitopes, or combinationsof two or more epitopes thereof.

In additional embodiments there are provided isolated polyepitopepolypeptides, which polypeptides comprise an amino acid sequenceselected from the group consisting of sequences provided in SEQ ID NOs:2, 4, 5, 6, 8 and 10. Also provided are mixtures of two or more isolatedpolyepitope polypeptides, including (but not limited to) mixtures of thepolypeptides having sequences as shown in SEQ ID NOs: 2 and 8, 2 and 10,4 and 8, 4 and 10, 5 and 8, 5 and 10, 6 and 8, and 6 and 10.

Other embodiments are isolated polynucleotides (nucleic acid molecules)which encode one of the polyepitope polypeptides described herein.Specific examples of such nucleic acid molecules comprise a sequenceselected from the group consisting of sequences recited in SEQ ID NOs:1, 3, 7, 9 and complements thereof. Other specific examples of nucleicacid molecules are the portions of each of SEQ ID NOs: 1, 3, 7, and 9which correspond to and encode the polyepitope polypeptides shown in SEQID NOs: 2, 4, 8, and 10, respectively.

Also provided herein are genetic constructs that comprise at least onenucleic acid molecule encoding a polyepitope polypeptide, and host cellstransformed with such a genetic construct.

Yet another embodiment is a composition comprising at least onepolyepitope polypeptide or at least one nucleic acid molecule encoding apolyepitope polypeptide, and at least one component selected from thegroup consisting of pharmaceutically acceptable carriers and adjuvants.This disclosure further provides methods for eliciting and/or enhancingan immune response in a subject, which methods involve administering tothe subject such a composition. In one specific, non-limiting example,the subject is infected with HIV-1

IV. Multi-Clade, Multivalent HIV-1 Constructs

The current disclosure provides multi-clade multivalent HIV-1 constructsuseful for inducing immune responses in HIV-1-infected populations withdiverse HLA alleles and HIV subtypes.

HIV-1 MCMV constructs comprise synthetic nucleic acid sequences to beused as HIV-1 immune-stimulatory and/or vaccine constructs to protectagainst multiple HIV-1 subtypes. These synthetic nucleic acid moleculesor mixtures of nucleic acid molecules are designed to elicit both T-celland B-cell responses against highly conserved epitopes within multipleHIV-1 subtypes. In specific embodiments, the synthetic genes arecontained in plasmid constructs, which can be used as a DNA vaccine, aswell as a source of recombinant protein for subsequent protein boosts.

Provided herein in various embodiments are multi-clade, multivalentpolyepitope polypeptides, which are useful to induce immunogenicresponses in vertebrate animals to HIV-1. These polypeptides includeCTL-stimulatory epitopes, T-helper cell stimulatory epitopes, B-cellstimulatory epitopes, or combinations of two or more such types ofepitopes. Epitopes in the polyepitope polypeptides are selected toprovide multi-clade coverage. In particular, epitopes are selected to beat least 50% conserved across a plurality of HIV-1 subtypes, forinstance, at least 2, 3, 4, 5, 6, or more HIV-1 subtypes. In particularembodiments, at least 30% of the epitopes included in a singlepolyepitope polypeptide are at least 60% conserved, at least 70%conserved, at least 80% conserved, or even more highly conserved acrossa plurality of HIV-1 subtypes.

One aspect of embodiments provided herein is that peptide sequences,each of which contains one or more antibody-binding or class I or classII MHC-binding epitopes, can be linked in tandem to form polyepitopepolypeptides. These polyepitope polypeptides are proteolyticallyprocessed in cells to release the individual epitopes, and are usefulfor stimulating an immune response in a vertebrate animal.

When a MCMV polyepitope polypeptide is introduced into a cell, it isproteolytically processed into at least some of its constituentepitopes. At least some of the epitopes generated from the polypeptidecan bind to MHC class I or MHC class II molecules present in the cell,though some of the epitopes may be specific for MHC class I or class IImolecules present only on other cells. Included epitopes also may beB-cell epitopes, which elicit antibody-mediated immune responses uponbinding to antibody receptors on the surface of a B-cell.

In one aspect, the disclosure features a nucleic acid encoding apolyepitope polypeptide that can include, in any order, a first, second,and third segment, each of which is at least nine amino acids in length.As used herein, a “segment” is an amino acid sequence which (a)corresponds to the sequence of a portion (that is, a fragment, less thanall) of a naturally occurring protein, and (b) contains one or moreepitopes. By “epitope” is meant a peptide which binds to the bindinggroove of an MHC class I or class II molecule, or to the antigen-bindingregion of an antibody. In addition, the polyepitope polypeptide canencode a targeting signal, for instance a peptide sequence that targetsthe protein to which it is fused to the lysosome or to the proteosome,as described in more detail herein.

In some embodiments, a segment of the polyepitope polypeptide has theamino acid sequence of a portion of (1) a naturally occurring HIV-1protein or (2) a naturally occurring coreceptor (collectively referredto as “naturally occurring proteins”), that is at least nine amino acidsin length. A second segment has the amino acid sequence of a secondportion of the same or a different naturally occurring protein, is atleast nine amino acids in length, and includes at least one epitopewhich is different from the epitope(s) of the first segment. Optionally,a third segment is included in the polyepitope polypeptide, and has theamino acid sequence of a third portion of the same or a differentnaturally occurring protein, is at least nine amino acids in length, andincludes at least one epitope which is different from the epitope(s) ofthe first and second segments. Alternatively, the first, second andthird portions may be portions of two or three different naturallyoccurring proteins (for example, two or three different HIV-1 proteins).The polyepitope polypeptide may optionally include additional segments,for example, it can include at least 4, 5, 10, 15, 20, 25, 30, 40, 50,60, 75, 90 or even 100 or more segments, each being a portion of anaturally-occurring protein of a pathogenic agent and/or of a naturallyoccurring coreceptor which can be the same or different from theprotein(s) from which the other segments are derived.

Each of these segments is at least nine amino acids in length, and eachcontains at least one epitope different from the epitope(s) of othersegments in the polyepitope polypeptide. At least one (and in someembodiments, more) of the segments in the polyepitope polypeptide maycontain class I or class II MHC-binding epitopes. Two, three, or more ofthe segments can be contiguous in the polyepitope polypeptide: that is,they are joined end-to-end, with no spacer between them. Alternatively,any two adjacent segments can be linked by a spacer amino acid or spacerpeptide. In particular embodiments, the spacer comprises three aminoacids. Specific non-limiting examples of spacers are the tri-amino acidKAA and the tri-amino acid PGP. Additionally, a spacer amino acid orspacer peptide can be used to link multiple groups of two, three, ormore contiguous segments in the polyepitope polypeptide: that is, aspacer amino acid or spacer peptide is inserted between every two,three, or more segments.

A given segment of protein within the polyepitope polypeptide need notbe any specified length, so long as it is sufficiently long to generateat least one epitope, for example, 2, 3, 4, 5, or more epitopes, and isat least 9 amino acids in length. For example, a given segment can havea length of at least 10 amino acids, for example, at least 11, 12, 13,14, 15, 20, 25, 30, 40, or 50 amino acids. A given segment correspondsto a particular naturally occurring protein if any 9 (or more)consecutive amino acids of the segment are found in exactly the sameorder in a portion of the naturally occurring protein. In exemplaryembodiments, the segments included in a polyepitope polypeptide areobtained from one or more HIV-1 proteins and/or coreceptors (forexample, CCR5).

It is understood that the term “naturally occurring proteins” used abovedoes not preclude modification of the sequence used in the polyepitopepolypeptide, for instance by changing one or a few amino acids. Inaddition, it is understood that the nucleic acid molecule encoding thesegment need not be identical to the “naturally occurring” sequence, asfound in (for instance) the HV-1 genome. In particular, it iscontemplated that the codon usage in the nucleic acid molecule can bemodified, for instance to convert the encoding sequence to a codonoptimized sequence. The codon optimization can be tailored for the hostcell in which the construct will eventually be expressed. Thus, someconstructs are engineered to be codon biased for expression in aprokaryotic cell, others to be expressed in a unicellular eukaryoticcell, and still others to be expressed in a cell of a multicellulareukaryote (for example, a vertebrate). Codon selection to take advantageof species biases is well known to those of ordinary skill.

The discovery of the HIV-1 coreceptors, together with a greaterunderstanding of the Envelope-receptor mediated conformational changesresulting in the membrane fusion process, has identified severalpromising vaccine targets. These epitopes as well as others in thetransmembrane envelope glycoprotein (gp41) have been identified as HIV-1neutralizing epitopes. Likewise, epitopes in the CCR5 coreceptor havebeen identified as potential targets for interfering with receptor-envinteractions. Any of these epitopes can be included in the polyepitopepolypeptides described herein.

Construction of HIV-MCMV Immunogens

HIV-1-MCMV immunogen constructs comprised of a string of codon-optimizedepitopes have been produced. The antigenic fragments/epitopes inexamples of such constructs were selected using published studiesincluding broad MHC allele recognition and were compiled from the LosAlamos sequence database. A representative pair of immunogen constructs(polyepitope polypeptides) contains multiple B-cell epitopes, CTLepitopes, and T-helper epitopes representing immunodominant regions forall subtypes of HIV-1 (see tables included in the examples, and FIGS. 1,2, and 3). The epitopes chosen are >80% homologous across diverse HIV-1subtypes. B-cell epitopes in the virus binding domain of the human HIVcoreceptor CCR5 are also included.

Without intending to be limited to a single interpretation, it isbelieved that antibodies to CCR5 together with neutralizing antibodiesdirected against the HIV-1 envelope glycoprotein and strong T-cellimmunity will interfere with the viral entry process and is expected toinduce sterilizing immunity.

Example immunogen constructs are shown in SEQ ID NOs: 2, 4, 5, 6, 8, and10. The constructs shown in SEQ ID NOs: 2, 4, 5, and 6 include CTLepitopes (and therefore can be referred to generally as MCMV-CTLconstructs); those in SEQ ID NOs: 8 and 10 include B-cell and T-helperepitopes (and therefore can be referred to generally as MCMV-AB/Thconstructs).

Unique restriction enzyme digestion sites have been included in thenucleic acid constructs encoding the provided polyepitope polypeptides.These facilitate addition/deletion of epitopes, as well as the shuttlingof the polyepitope cassette between a number of DNA vectors, includingDNA vaccine constructs (for example, pVax-1, Invitrogen, Carlsbad,Calif.), eukaryotic yeast expression vectors (for example, pYes,Invitrogen, Carlsbad, Calif.), and multi-cell type expression vectors(for example, pTriEX-4, Novagen, Madison, Wis.). This enables theproduction of both a DNA based immunogen and vaccine, and readyproduction recombinant polyepitope polypeptide, which can be useddirectly as an immunogen or as a boost. The synthetic genes (whichencode one or more polyepitope polypeptides) also can be incorporatedinto attenuated viral vectors such as Modified Vaccinia or Adenovirus toserve as a boosting agent.

Delivery and Immunogenicity by Inclusion of Targeting Sequences

Recent studies suggest that peptide spacers between epitopes and/ortargeting sequences may increase the immunogenicity of certain epitopes.Targeting sequences such as the LIMP-II targeting sequence (whichdirects proteins to lysosomes and enhances class-II recognition), ortargeting-competent fragments thereof, are used in certain providedembodiments to help enhance T-helper response. Likewise, proteosometargeting sequences (for example, ubiquitin or targeting-competentfragments thereof) that help induce class I recognition are included inspecific embodiments, to provide improved CTL production. The chosenepitopes were back translated and human codon optimized for increasedexpression from the DNA construct.

In any of the described nucleic acids encoding polyepitope polypeptides,a spacer amino acid or spacer peptide can be included between any twoadjacent segments of the construct. Optionally, in some embodiments thespacer is included between each epitope; in other embodiments, a spaceris included between every two, every three, every four, every fiveepitopes, or even less often. In particular embodiments, the spacercomprises three amino acids. Specific non-limiting examples of spacersare the tri-amino acid KAA and the tri-amino acid PGP.

Recognition of Epitopes Contained in the Constructs

Most vaccine constructs under development are subtype-specific. This hasled to development of a number of country-specific subtype-specificHIV-1 vaccines, however, such vaccines will be difficult to implementdue to emerging diversity and changing epidemic of HIV-1.

In contrast, the constructs provided herein comprise highly conservedimmunogenic regions of HIV-1 that result in cross-protective immuneresponses across HIV-1 subtypes. The immune responses to the immunogenicepitopes can be tested, for instance, in recently-infected HIV-1infected persons (Primary HIV-1 infection; PHI) or individuals that havea slow progression to disease.

V. Uses of MCMV Immunogens

In order to function effectively in vivo as a DNA-based immunogen, it isadvantageous to include within the MCMV nucleic acid construct a controlsequence that has the effect of enhancing or promoting the translationof the sequences encoding the antigens. Use of such promoters is wellknown to those of skill in the fields of molecular biology, cellbiology, and viral immunology (See, “Molecular Cloning: A LaboratoryManual”, 2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989; and “Current Protocols inMolecular Biology”, Ausubel et al., John Wiley and Sons, New York 1987(updated quarterly)).

In certain embodiments, the nucleic acid construct is intended for useas a vaccine in a mammalian host. Therefore it is advantageous to employa promoter which operates effectively in mammalian cells. Particularembodiments relate to both prokaryotic and eukaryotic host cells. Manypromoter sequences are known that are useful in either prokaryotic oreukaryotic host cells. A promoter is operably disposed with respect tothe sequence(s) whose translation is to be promoted, so that it iscapable of promoting translation. In certain embodiments, the promoteris the cytomegalovirus early promoter. In addition, in some embodiments,the sequences to be expressed are followed by a terminator sequence.

Preparation of the nucleic acids is readily accomplished by methods wellknown to workers of skill in the field of molecular biology. Proceduresinvolved are set forth, for example, in Sambrook, Fritsch and Maniatis,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, and“Current Protocols in Molecular Biology”, Ausubel et al., John Wiley andSons, New York 1987 (updated quarterly). Incorporation of promoters,such as the cytomegalovirus promoter, and of the polyadenylation signal,is likewise well known to skilled practitioners in molecular biology andrecombinant DNA engineering.

When a nucleic acid molecule harboring a MCMV epitope chain is prepared,it may be obtained in larger quantities by methods that amplify anucleic acid fragment. Such methods are widely known to workers skilledin molecular biology and recombinant DNA engineering. Examples of thesemethods include incorporation of the nucleic acid fragment into aplasmid for replication by culturing in a cell (for example, aprokaryotic cell) and harvesting the plasmid after growth of theculture, as well as amplification of the nucleic acid fragment bynucleic acid amplification methods, such as the PCR. These methods areexemplary only, and not intended to limit the ways in which the nucleicacid construct may be obtained.

The MCMV nucleic acid constructs may be introduced into appropriate hostcells in many ways well known to those of ordinary skill in the fieldsof molecular biology and viral immunology. By way of example, theseinclude, but are not limited to, incorporation into a plasmid or similarnucleic acid vector which is taken up by the host cells, orencapsulation within vesicular lipid structures such as liposomes,especially liposomes comprising cationic lipids, or adsorption toparticles that are incorporated into the host cell by endocytosis.

In general, a host cell is a prokaryotic or eukaryotic cell harboring aMCMV nucleic acid, or into which such a MCMV molecule has beenintroduced. The constructs described herein induce the intracellularbiosynthesis of the encoded multivalent HIV-1 antigens. A suitable hostcell is one which has the capability for the biosynthesis of the geneproducts as a consequence of the introduction of the nucleic acid. Inparticular embodiments, a suitable host cell is one which responds to acontrol sequence and to a terminator sequence, if any, which may beincluded within the construct. In order to respond in this fashion, sucha host cell contains within it components which interact with a controlsequence and with a terminator, and act to carry out the respectivepromoting and terminating functions. When the host cell is cultured invitro, it may be a prokaryote, a single-celled eukaryote or a vertebratecell. In particular embodiments, the host cell is a mammalian cell

VI. Stimulation of Immunological Responses to HIV-1

With the provision herein of polyepitope polypeptide antigens specificto HIV-1, methods are now enabled for the stimulation of immuneresponses to such antigens in subjects. In certain embodiments, suchimmune responses will be protective against HIV-1 infection in thesubject. MCMV polyepitope polypeptides (singly or in combination) can beused, for instance, as immunogenic agents in the inhibition, treatment,or amelioration of HIV-1. Subjects selected for this type of treatmentare those who are known to have, or are suspected of having or are atrisk of suffering, a HIV-1 infection.

The provided immunostimulatory MCMV polyepitope polypeptides, orconstructs or vectors encoding such polypeptides, are combined with apharmaceutically acceptable carrier or vehicle for administration as animmunostimulatory composition or a vaccine to human or animal subjects.In some embodiments, more than one polyepitope polypeptide may becombined to form a single preparation.

The immunogenic formulations may be conveniently presented in unitdosage form and prepared using conventional pharmaceutical techniques.Such techniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers.Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, for example, water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets commonly used by oneof ordinary skill in the art.

In certain embodiments, unit dosage formulations are those containing adose or unit, or an appropriate fraction thereof, of the administeredingredient. It should be understood that in addition to the ingredientsparticularly mentioned above, formulations encompassed herein mayinclude other agents commonly used by one of ordinary skill in the art.

The compositions provided herein, including those for use asimmunostimulatory agents or vaccines, may be administered throughdifferent routes, such as oral, including buccal and sublingual, rectal,parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal,and topical. They may be administered in different forms, including butnot limited to solutions, emulsions and suspensions, microspheres,particles, microparticles, nanoparticles, and liposomes.

The volume of administration will vary depending on the route ofadministration. By way of example, intramuscular injections may rangefrom about 0.1 ml to about 1.0 ml. Those of ordinary skill in the artwill know appropriate volumes for different routes of administration.

The amount of protein in each vaccine dose is selected as an amount thatinduces an immunostimulatory or immunoprotective response withoutsignificant, adverse side effects. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented. Initialinjections may range from about 1 μg to about 1 mg, with someembodiments having a range of about 10 μg to about 800 μg, and stillother embodiments a range of from about 25 μg to about 500 μg. Followingan initial vaccination, subjects may receive one or several boosterimmunizations, adequately spaced. Booster injections may range fromabout 1 μg to about 1 mg, with other embodiments having a range of about10 μg to about 750 μg, and still others a range of about 50 μg to about500 μg. Periodic boosters at intervals of 1-5 years, for instance threeyears, may be desirable to maintain the desired levels of protectiveimmunity.

As described in WO 95/01441, the course of the immunization may befollowed by in vitro proliferation assays of PBL (peripheral bloodlymphocytes) co-cultured with ESAT6 or ST-CF, and especially bymeasuring the levels of IFN-released from the primed lymphocytes. Theassays are well known and are widely described in the literature,including in U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064.

A relatively recent development in the field of immune stimulatorycompounds (for example, vaccines) is the direct injection of nucleicacid molecules encoding peptide antigens (broadly described in Janeway &Travers, Immunobiology: The Immune System In Health and Disease, page13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari,N. Engl. J. Med. 334:42-45, 1996). Plasmids (vectors) that includenucleic acid molecules described herein, or that include a nucleic acidsequence encoding an immunogenic MCMV polyepitope polypeptide may beutilized in such DNA vaccination methods.

Thus, the terms “immunostimulatory preparation” and “vaccine” as usedherein also include nucleic acid vaccines in which a nucleic acidmolecule encoding a MCMV polyepitope polypeptide is administered to asubject in a pharmaceutical composition. For genetic immunization,suitable delivery methods known to those skilled in the art includedirect injection of plasmid DNA into muscles (Wolff et al., Hum. Mol.Genet. 1:363, 1992), delivery of DNA complexed with specific proteincarriers (Wu et al., J. Biol. Chem. 264:16985, 1989), co-precipitationof DNA with calcium phosphate (Benvenisty and Reshef, Proc. Natl. Acad.Sci. 83:9551, 1986), encapsulation of DNA in liposomes (Kaneda et al.,Science 243:375, 1989), particle bombardment (Tang et al., Nature356:152, 1992; Eisenbraun et al., DNA Cell Biol. 12:791, 1993), and invivo infection using cloned retroviral vectors (Seeger et al., Proc.Natl. Acad. Sci. 81:5849, 1984).

Similarly, nucleic acid vaccine preparations can be administered viaviral carrier.

It is also contemplated that the provided immunostimulatory moleculesand preparations can be administered to a subject indirectly, by firststimulating a cell in vitro, which stimulated cell is thereafteradministered to the subject to elicit an immune response.

VII. Immunological and Pharmaceutical Compositions

Immunological compositions, including immunological elicitorcompositions and vaccines, and other pharmaceutical compositionscontaining latency-specific polypeptides or antigenic fragments thereofare useful for reducing, ameliorating, treating, or possibly preventingHIV infection, particularly HIV-1 infection. One or more of thepolypeptides are formulated and packaged, alone or in combination withadjuvants or other antigens, using methods and materials known to thoseskilled in the vaccine art. An immunological response of a subject tosuch an immunological composition may be used therapeutically orprophylactically, and in certain embodiments provides antibody immunityand/or cellular immunity such as that produced by T-lymphocytes, such ascytotoxic T-lymphocytes or CD4⁺ T-lymphocytes.

The MCMV polyepitope polypeptides may be administered with an adjuvantin an amount effective to enhance the immunogenic response against theconjugate. At this time, the only adjuvant widely used in humans hasbeen alum (aluminum phosphate or aluminum hydroxide). Saponin and itspurified component Quil A, Freund's complete adjuvant and otheradjuvants used in research and veterinary applications have toxicitieswhich limit their potential use in human vaccines. However, chemicallydefined preparations such as muramyl dipeptide, monophosphoryl lipid A,phospholipid conjugates such as those described by Goodman-Snitkoff etal. (J. Immunol 147:410415, 1991), encapsulation of the conjugate withina proteoliposome as described by Miller et al. (J. Exp. Med176:1739-1744, 1992), and encapsulation of the protein in lipid vesiclesmay also be useful.

The compositions provided herein, including those formulated to serve asvaccines, may be stored at temperatures of from about −100° C. to about4° C. They may also be stored in a lyophilized state at differenttemperatures, including higher temperatures such as room temperature.The preparation may be sterilized through conventional means known toone of ordinary skill in the art. Such means include, but are notlimited to, filtration, radiation and heat. The preparations also may becombined with bacteriostatic agents, such as thimerosal(ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Co.,St. Louis, Mo.), to inhibit bacterial growth.

A variety of adjuvants known to one of ordinary skill in the art may beadministered in conjunction with the protein(s) in the provided vaccinecomposition. Such adjuvants include but are not limited to thefollowing: polymers, co-polymers such aspolyoxyethylene-polyoxypropylene copolymers, including blockco-polymers; polymer P1005; Freund's complete adjuvant (for animals);Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300adjuvant; alum; QS 21, muramyl dipeptide; CpG oligonucleotide motifs andcombinations of CpG oligonucleotide motifs; trehalose; bacterialextracts, including mycobacterial extracts; detoxified endotoxins;membrane lipids; or combinations thereof.

In a particular embodiment, a vaccine is packaged in a single dosage forimmunization by parenteral (that is, intramuscular, intradermal orsubcutaneous) administration or nasopharyngeal (that is, intranasal)administration. In certain embodiments, the vaccine is injectedintramuscularly into the deltoid muscle. The vaccine may be combinedwith a pharmaceutically acceptable carrier to facilitate administration.The carrier is, for instance, water, or a buffered saline, with orwithout a preservative. The vaccine may be lyophilized for resuspensionat the time of administration or in solution.

The carrier to which the polypeptide may be conjugated may also be apolymeric delayed release system. Synthetic polymers are particularlyuseful in the formulation of a vaccine to affect the controlled releaseof antigens.

Microencapsulation of the polypeptide will also give a controlledrelease. A number of factors contribute to the selection of a particularpolymer for microencapsulation. The reproducibility of polymer synthesisand the microencapsulation process, the cost of the microencapsulationmaterials and process, the toxicological profile, the requirements forvariable release kinetics and the physicochemical compatibility of thepolymer and the antigens are all factors that must be considered.Examples of useful polymers are polycarbonates, polyesters,polyurethanes, polyorthoesters, polyamides,poly-(d,1-lactide-co-glycolide) (PLGA), and other biodegradablepolymers.

Doses for human administration of a pharmaceutical composition or avaccine may be from about 0.01 mg/kg to about 10 mg/kg, for instanceabout 1 mg/kg. Based on this range, equivalent dosages for heavier (orlighter) body weights can be determined. The dose may be adjusted tosuit the individual to whom the composition is administered, and mayvary with age, weight, and metabolism of the individual, as well as thehealth of the subject. Such determinations are left to the attendingphysician or another familiar with the subject and/or the specificsituation. The vaccine may additionally contain stabilizers orphysiologically acceptable preservatives, such as thimerosal.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES

Existing HIV-1 vaccine constructs are subtype specific. Though multiplesub-type specific candidate vaccines are under development, areas withhigh numbers of recombinant viruses would likely pose problems forsubtype specific vaccines.

While the high degree of HIV variability has made vaccine designdifficult, the proximity of populations with varying subtypes and theease of travel have made a vaccine that can provide protection frommultiple subtypes desirable. To address the problem of subtypevariability, these examples illustrate production of constructscontaining conserved B, T-helper, and CTL epitopes, with and withouttargeting domains. The chosen epitopes are expected to generate immuneresponses to multiple HIV-1 subtypes.

Selection of Epitopes

In order to assemble a set of CTL epitopes that were conserved across awide range of HIV-1 subtypes and that would be recognized by a largepercentage of the population, the following databases and sources ofsequence were consulted: Los Alamos HIV Molecular Immunology Database,Described Epitopes, LTNPs, EU and the Los Alamos HIV Sequence Database.The literature was also consulted, to locate reported conservedepitopes.

The epitopes were selected based on conserved epitopes previously shownto be recognized by HIV-1-infected persons from published reports or theLos Alamos Data base. These antigenic fragments/epitopes were selectedusing the results of in vitro and in vivo protection studies compiled inthe Los Alamos database, as well as using the Motifscan softwareprogram. The following table (Table 1) provides the list of the multipleepitopes in the highly conserved regions in gag (p17, p24) pol (Prt, RTand Int) as well as Nef, Vif, Vpr, and Env epitopes selected based ontheir MHC class I binding (CTLs).

Epitopes were selected that were greater than 50% conserved across allthe available sequences. Of these, the majority of epitopes were >90%conserved for subtypes A/B/C/D/E/F/G. Also taken into consideration wasthe frequency of Class I alleles that would recognize each conservedepitope. TABLE 1 AA Position in SEQ ID CTL Region Sequence NO: 2HLA-alleles Source p17 18-29 KIRLRPGGKKKY  96-107 A3, A3.1, B27, Ferrariet al. B42, Bw62 ARHR p17 36-44 WASRELERF 108-116 B35 16: 1433-1443, p1774-92 ELRSLYNTVATLYCVH 77-95 B8, A2, A11 2000 QRI p24 15-27ISPRTLNAWVKVV 120-132 A2, B57 p24 30-55 KAFSPEVIPMFSALSEG 133-158 B58,B44, B7 ATPQDLNTM p24 108-117 TSTLQEQIGW 159-168 B57 p24 121-150NPPIPVGDIYKRWIILG 172-201 B8, B35, Ferrari et al. LNKIVRMYSPTSI B27,B62, B52 ARHR p24 161-174 FRDYVDRFYKTLRA 202-215 B18, B44, A24, 16:1433-1443, B70, B14 2000, and Hanke et al., Nature Med 9: 951-955, 2000p24 191-205 VQNANPDCKTILKAL 216-230 B51, B8, Ferrari et al. 216-226ACQGVGGPGHK 231-241 A11 ARHR 16: 1433-1443, 2000 pol 59-65 ITLWQRPLV245-253 A28, A6802 Ferrari et al. ARHR 16: 1433-1443, 2000; Hanke etal., Nature Med 9: 951-955, 2000 pol 262-273 TVLDVGDAYFSV 254-265 A2,A0201, B35 Ferrari et al. ARHR 16: 1433-1443, 2000 pol 308-321WKGSPAIFQSSMTK N/A B7, B35, A11, Ferrari et al. (SEQ ID NO: 11) A3, A33ARHR 16: 1433-1443, 2000; Hanke et al., Nature Med 9: 951-955, 2000 pol464-472 ILKEPVHGV 308-316 A2, A0201 Ferrari et al. pol 495-507QIYQEPFKNLKTG 320-332 A11 ARHR pol 587-602 EPIVGAETFYVDGAAN 333-348 B35,B51, A28 16: 1433-1443, pol 956-964 LLWKGEGAV 358-366 A2, A0201 2000 polVIYQYMDDL 349-357 A0201 Hanke et al., Nature Med 9: 951-955, 2000 vif48-57 HPKVSSEVHI 380-389 B0702 Altfeld et al, J. Immunology 167:2743-2752 (non-B sequence was derived from sequence data contained inthe Los Alamos HIV sequence database Vif 17-26 RIRTWKSLVK 370-379 A0301Altfeld et al, J. vprB 29-42 AVRHFPRIWLHSL N/A B5701 Immunology (SEQ IDNO: 12) 167: 2743-2752 vpr 29-42 AVRHFPRPWLHGL N/A B7301* Altfeld et al,J. nonB (SEQ ID NO: 13) Immunology 167: 2743-2752 (non-B sequence wasderived from sequence data contained in the Los Alamos HIV sequencedatabase) vpr 58-66 AIIRILQQL 408-416 A0201 Altfeld et al, J. Immunology167: 2743-2752 nef 64-95 VGFPVRPQVPLRPMTY 420-451 A11, B8, B35, Ferrariet al. KGAVDLSHFLKEKGGL B7, A3, A2, ARHR 2000 nef 127-141 GPGVRYPLTFGWCY452-465 B57 16: 1433-1443, Hanke et al., Nature Med 9: 951-955, 2000gp120 36-51 TVYYGVPVWKEATTTL 478-493 A3, B35, B55 Ferrari et al. gp120120-128 KTLPLCVTL 469-477 A2 ARHR gp41 47-55 RAIEAQQHL 494-502 B51 16:1433-1443, 2000 gp41 ERYLKDQQL N/A B14, A24, B8 Hanke et al., (SEQ IDNO: 14) Nature Med SIV p27 ACTPYDINQML 291-301 MONKEY 9: 951-955,Mamu-A*01 2000 gp120 RGPGRAFVTI 278-287 Mouse H-2D

These epitopes can be further characterized in summary as follows: TABLE2 # of Epitope Epitopes HLA p17 6 A3, A3.1, B27, B42, Bw62, B35, B8, A2,A11 p24 20 B57, A2, B58, B44, B7, B57, B8, B35, B27, B2, B52, B18, B44,A24, B70, B14, B51, B8, B8, A11 pol 10 A28, B35, A2, A0201, B7, B35,A11, A3, A2, A0201, A11, B35, B51, A28, A2, A0201 nef 8 A1, B8, B35, B7,B35, A3, A11, A2, B35, A11, B8, B57 gp120 4 A3, B55, B35, A2 gp-41 2B51, B14 Vif 2 B0702, A0301 Vpr 3 B5701, B7301, A0201 SIVp27/gp120¹ 2MAMU-A*01/Mouse H-2D¹Control epitopes included for animal studies; SIVp27 is a simianepitope; the gp120 epitope is known to be recognized by the murine H-2DHLA.CTL/Proteosome Constructs

FIG. 1A shows the schematic map of one synthetic construct prepared toencode the identified conserved CTL epitopes. Representative nucleotideand amino acid sequences of HIV-1-MCMV-CTL with and without ubiquitinare shown in the accompanying Sequence Listing.

Using the 55 conserved CTL epitopes identified as described above, asynthetic gene was constructed using SOE followed by PCR. The synthesisis illustrated in FIG. 2A. A tri-amino acid spacer (KAA) was insertedbetween every 3-5 epitopes, to enhance peptide processing. Two parallelconstructs were constructed, one with and one without ubiquitin(included to provide proteosomal targeting and further enhance peptideprocessing). pVAXMCMV-CTL (FIG. 2C) contains a 1.3 KB fragment codingfor 55 CTL epitopes. pVAXMCMV-CTL-ubiquitin (FIG. 2B) contains a 1.5 KBfragment coding for 55 CTL epitopes, covalently linked (5′) to amodified ubiquitin molecule.

Expression of Multivalent Polypeptide from a MCMV Construct

Recombinant proteins encoded by the synthetic genes have been producedin E. coli (FIG. 4). The expressed protein is larger, because the fusiontag attached to the protein is large. The fusion is detected (forexample, in FIG. 4) with an anti-Histidine monoclonal antibody, whichdetects the fusion tag (poly-His) encoded by the vector.

Protein expression of Ubiquitin-CTL gene also has been confirmed in HeLacells. A typical western blot analysis of HeLa Cells transfected with 1μg of pVAX MCMV-CTL-ubiquitin is shown in FIG. 5. An anti-ubiquitinantibody was used for detection. These results indicate expression of anubiquitinated protein of the correct predicted molecular weight as thesynthetic CTL-ubiquitin gene. A band representing the normal cellularubiquitin protein is also present on the blot.

Elispot Assays

To determine the biological relevance of epitopes used in the MCMV-CTLconstruct, epitopes contained in the construct were tested forrecognition with PBMCs from HIV-1 infected individuals by doing Elispotassays. This example demonstrates that people infected with geneticallydifferent viruses could recognize epitopes included in the construct.Also since the individuals being tested are from different geographiclocations, they are expected to have differences in their MHC molecules.

Epitope Testing (Elispot)

Chronically HIV-1-infected individuals infected with subtype B wereselected from the HIV outpatient clinic at Johns Hopkins Hospital(Baltimore, Md.) for testing cellular immune responses to HIV-1 (Keatinget al., AIDS Research and Human Retroviruses 18:1067-1079, 2002).Subject median age was 42 years, with a range of 25-58 years. Medianviral load was 2,228 copies/ml with a range of less than 330 copies/mlto 37,716 copies/ml. Two of the subjects (Nos. 10 and 15) used in theanalysis had viral loads of greater than 15,000 copies/ml, and wereidentified as non-responders to subtype B Gag peptides in the test ofKeating et al. (AIDS Research and Human Retroviruses 18:1067-1079,2002). The average number of years patients were HIV-1 infected was 9.46years (range 5-17 years), and median CD4+ cell count was 534 cells/mm³with a range of 294-1009 cells/mm³. These data represent a unique cohortof patients with strong immunological control as they have high CD4values and have been HIV-1 infected for an average of nine years.

Peripheral blood was obtained by venipuncture and collected in heparin(Sigma, St. Louis, Mo.). PBMCs were isolated by Ficoll-Hypaque(Pharmacia-Amersham, Piscataway, N.J.) gradient centrifugation, frozenin fetal calf serum (FCS; Summit) with 10% DMSO (Sigma, St. Louis, Mo.)and stored at −140° C.

A 9×9 matrix (shown below) representing the 55 epitopes contained in theconstruct plus 23 control peptides from Flu, EBV and CMV was generatedto study Elispot responses, using 1×10⁵ cryopreserved PMBCs per well and5 μg/ml peptide (below). Samples were run in duplicate, and the positivecutoff defined as wells that had 2× more spots than negative controlwells and at least 10 spots. Responses identified in the matrix testingwere confirmed with individual peptide testing. Elispot was carried outessentially as described in Keating et al., AIDS Research and HumanRetroviruses 18:1067-1079, 2002. TABLE 3 Peptide matrix M1 M2 M3 M4 M5M6 M7 M8 M9 MA  1-p17  2-p17  3-p17  4-p17  5-p17  6-p17  7-p24  8-p24 9-p24 MB 10-p24 11-p24 12-p24 13-p24 14-p24 15-p24 16-p24 17-p24 18-p24MC 19-p24 20-p24 21-p24 22-p24 23-p24 24-p24 25-p24 26-p24 27-pol MD28-pol 29-pol 30-pol 31-pol 32-pol 33-pol 34-pol 35-pol 36-pol ME 37-nef38-nef 39-nef 40-nef 41-nef 42-nef 43-nef 44-nef 45- gp120 MF 46- 47-48- 49-gp41 50-gp41 51-vif 52-vif 53-vpr 54-vpr gp120 gp120 gp120 MG55-vpr 56-flu 57-flu 58-EBV 59-flu 60-CMV 61-flu 62-EBV 63-EBV MH 64-EBV65-EBV 66-EBV 67-Flu 68-EBV 69-CMV 70-EBV 71-EBV 72-EBV MI 73-Flu 74-Flu75-EBV 76-EBV 77-EBV 78-CMV

TABLE 4 Peptides in the Matrix AA Position in SEQ No. Peptide listSequences ID NO: 2 HLA 1 p17-1 KIRLRPGGK  96-104 A3, A3.1, B27 2 p17-2RLRPGGKKKY  98-107 B42, Bw62 3 p17-3 WASRELERF 108-116 B35 4 p17-4ELRSLYNTV 77-85 B8 5 p17-5 SLYNTVATL 80-88 A2 6 p17-6 TLYCVHQRI 87-95A11 7 p24-1 ISPRTLNAW 120-128 B57 8 p24-2 TLNAWVKVV 124-132 A2 9 p24-3KAFSPEVIPMF 133-143 B58 10 p24-4 IPMFSALSEGATPDQL N/A B44 (SEQ ID NO:15) 11 p24-5 ATPQDLNTM 150-158 B7 12 p24-6 TSTLQEQIGW 159-168 B57 13p24-7 NPPIPVGEIYKRWII N/A B8 (SEQ ID NO: 16) 14 p24-8 PPIPVGDIY 173-181B35 15 p24-9 KRWIILGLNKIV 182-193 B27 16 p24-10 LGLNKIVRMYS 187-197 B6217 p24-11 RMYSPTSI 194-201 B52 18 p24-12 FRDYVDRFYK 202-211 B18 19p24-13 RDYVDRFYKTL 203-213 B44 20 p24-14 DYVDRFYKTL 204-213 A24 21p24-15 YVDRFYKTL 205-213 B70 22 p24-16 DRFYKTLRA 207-215 B14 23 p24-17VQNANPDCKTILKAL 216-230 B51 24 p24-18 NANPDCKTI 218-226 B8 25 p24-19DCKTILKAL 222-230 B8 26 p24-20 ACQGVGGPGHK 231-241 A11 27 POL-1ITLWQRPLV 245-253 A28 28 POL-2 TVLDVGDAY 254-262 B35 29 POL-3VLDVGDAYFSV 255-265 A2, A0201 30 POL-4 WKGSPAIFQSSMT N/A B7, B35 (SEQ IDNO: 17) 31 POL-5 AIFQSSMTK (SEQ ID NO: 18) N/A A11, A3 32 POL-6ILKEPVHGV 308-316 A2, A0201 33 POL-7 QIYQEPFKNLKTG 320-332 A11 34 POL-8EPIVGAETF 333-341 B35, B51 35 POL-9 AETFYVDGAAN 338-348 A28 36 POL-10LLWKGEGAV 358-366 A2, A0201 37 NEF-1 VGFPVTPQVPLRPMT N/A A1, B8 (SEQ IDNO: 19) 38 NEF-2 FPVRPQVPL 422-430 B35 39 NEF-3 FPVRPQVPLR 422-431 B7 40NEF-4 RPQVPLRPMTY 425-435 B35 41 NEF-5 QVPLRPMTYK 427-436 A3, A11, A2,B35 42 NEF-6 AVDLSHFLK 438-446 A11 43 NEF-7 FLKEKGGL 444-451 B8 44 NEF-8GPGVRYPLTFGWCY 452-465 B57 45 gp120-1 TVYYGVPVWK 478-487 A3 46 gp120-2VPVWKEATTT 483-492 B55, 47 gp120-3 VPVWKEATTTL 483-493 B35 48 gp120-4KTLPLCVTL 469-477 A2 49 gp-41-1 RAIEAQQHL 494-502 B51 50 gp-41-1ERYLKDGGL 503-511 B14 51 VIF-1 HPKVSSEVHI 380-389 B0702 52 VIF-2RIRTTWKSLVK N/A A0301 (SEQ ID NO: 20) 53 VPRB-1 AVRHFPRIWLHSL N/A B5701(SEQ ID NO: 21) 54 VPRNB-2 AVRHFPRPWLHGL N/A B7301 (SEQ ID NO: 22) 55VPR-3 AIIRILQQL 408-416 A0201 56 Influenza A PB1 591-599 VSDGGPNLY N/AA1 (SEQ ID NO: 23) 57 Influenza A NP 44-52 CTELKLSDY (SEQ ID NO: 24) N/AA1 58 EBV BMLF 259-267 GLCTLVAML N/A A2 (SEQ ID NO: 25) 59 Influenza AMatrix 1 58-66 GILGFVFTL (SEQ ID NO: 26) N/A A2 60 HCMV Pp65 495-503NLVPMVATV N/A A2 (SEQ ID NO: 27) 61 Influenza A NP 265-273 ILRGSVAHK(SEQ ID NO: 28) N/A A3 62 EBV BMLF 259-267 RVRAYTYSK N/A A3 (SEQ ID NO:29) 63 EBV EBNA3A 603-611 RLRAEAQVK N/A A3 (SEQ ID NO: 30) 64 EBV EBNA3B416-424 IVTDFSVIK (SEQ ID NO: 31) N/A A11 65 EBV BRLF1 134-143 ATIGTAMYKN/A A11 (SEQ ID NO: 32) 66 EBV BRLF1 28-37 DYCNVLNKEF N/A A24 (SEQ IDNO: 33) 67 Influenza A NP 91-99 KTGGPIYKR (SEQ ID NO: 34) N/A Aw68 68EBV EBNA3A 379-387 RPPIFIRRL (SEQ ID NO: 35) N/A B7 69 HCMV Pp65 495-503TPRVTGGGAM N/A B7 (SEQ ID NO: 36) 70 EBV EBNA3A 158-166 QAKWRLQTL N/A B8(SEQ ID NO: 37) 71 EBV EBNA3A 325-333 FLRGRAYGL N/A B8 (SEQ ID NO: 38)72 EBV BZLF1 190-197 RAKFKQLL (SEQ ID NO: 39) N/A B8 73 Influenza A NP380-388 ELRSRYWAI (SEQ ID NO 40) N/A B8 74 Influenza A NP 380-388SRYWAIRTR (SEQ ID NO: 41) N/A B27 75 EBV EBNA3C 258-266 RRIYDLIEL (SEQID NO: 42) N/A B27 76 EBV EBNA3A 458-466 YPLHEQHGM N/A B35 (SEQ ID NO:43) 77 EBV EBNA3C 281-290 EENLLDFVRF N/A B44 (SEQ ID NO: 44) 78 HCMVPp65 495-503 QEFFWDANDIYRIFA N/A B44 (SEQ ID NO: 45)Results:

Eleven individuals chronically infected with HIV-1 subtype B (thesubtype found in the US) were tested. Preliminary testing of individualsform Ivory Coast, West Africa (subtype A/G viruses) was also conducted.These data indicate that the CTL epitopes contained in the construct arerecognized by individuals infected with genetically distinct subtypes ofHIV-1.

The eleven patients had confirmed responses to multiple epitopes, inp17, p24, pol, vpr, gp41, gp120, and nef Representative bar graphsdemonstrating breadth and magnitude of CTL responses generated fromPBMCs of six of the eleven individuals chronically infected withsubtype/clade B HIV-1 are shown in FIG. 6. These patient data are alsosummarized in the following table (which also includes data from theremaining five individuals), and indicate the viral load of theindividual, their CD4 count, the known HLA type of the individual andthe known HLA binding properties of the epitopes to which the individualresponded. TABLE 5 Patient # CD4 # Viral load Patient HLA Responseregions Epitope HLA  3 501 11,694 A30, A33, B53, B14 p24-6, Nef-8 B57  7349 6,177 NA Pol-6 A2, A02201 10 700 37,716 NA Pol-9 A28 11 924 1742A33, A68, B7, B57 p24-1, p24-3, p24-4, B57, B58, B44, VprB-1, Pol-9 B35,B5701 14 843 <50 A2, A30, B13, B27 p24-9, VprNB-2 B27, B7301 15 34920,354 NA EBV Control only B35 16 434 2714 A3, A26, B49, B65 p17-1,p24-16 A3, B27, B14 18 493 5862 A2, A29, B14, B72 p17-5, p24-5, p24-16A2, B7, B14 19 822 1490 NA p24-1, p24-3 B57, B58 21 567 <29 A3, A23,B35, B62 P17-1, p17-5 Nef-8 A3, B27, A2, gp41-1, gp41-2 B57, B14 22 1009<50 A23, B57, B72 p24-1, p24-3, p24-6, B57, B58, B57, Nef-8, gp120-1 B57Neg NA Uninfected NA EBV Control only B8 Control

Ten of the eleven (10/11) patients (90.9%) responded to one or morepeptides; 8/11 (72.7%) responded to two or more peptides in themultivalent construct. A summary of the peptides recognized from eachgene region by the 11 chronically infected individuals (Subtype B) isprovided in Table 6. TABLE 6 Peptides Gene Region Recognized PercentageAll peptides 18/55 32.7 Gag 10/26 38.4 Pol  2/10 20 Nef 1/8 12.5 Env 3/650 Vif 0/2 0 Vpr 2/3 66

Of the seven chronically HIV-infected study subjects who responded toHIV-1 individual peptides and had HLA typing made available, HLAspecificities of the CTL epitopes were compared with the patient HLAtypes. All seven individuals responded to at least one of the predictedCTL epitopes according to the restricting HLA allele of that epitope.There was great variability as to whether patient cells could targetthose CTL epitopes predicted. FIG. 7 shows the percentage of predictedepitopes that were targeted by the patients' CD8⁺ cells in the Elispotassay. Patient number 21 responded to only one of the 12 peptidespredicted to be targeted according to the patient's HLA type, whereaspatient number 22 recognized all three of the peptides predicted toelicit Elispot responses. All of the 7 subject studies were capable oftargeting epitopes outside of their respective HLA type. For example,patient number 3 who had been characterized for A30, A33, B53, and B14HLA type, only recognized p24 peptide sequence TSTLQEQIGW, which is aB57 HLA restricted CTL epitope. Of the 7 peptides targeted by patientnumber 11, four were epitopes restricted to mismatched HLA haplotype.All five of the HLA restricted epitopes targeted by patient number 21were disparate for that individual's HLA type.

Preliminary testing of individuals infected with other subtypes of virus(A from Kenya, C from India and A/E from Thailand) is underway.

Additional testing was carried out using PBMCs from HIV-1 positive blooddonors from Ivory Coast. The testing was done as described for thesubtype B individuals, in that the same peptide pool matrix was testedas describe above. The four Ivorian samples tested were from “healthy”blood donors and thus were presumed to be incident HIV-1 infections. Dueto the high prevalence of recombinant subtype A/G viruses in thisregion, it is presumed these individuals were infected with A/G viruses.The four individuals had broad responses based on the results of thepeptide matrix screen.

Individual 1 had predicted responses from the matrix to 46 peptidescontained in MCMV. The gene regions represented by these peptidesincluded p24, pol, nef, gp120, vpr, and vif. Individual 2 had predictedresponses to 15 peptides (in pol, nef, gp41, and vif). Individual 3 hadpredicted responses to 10 peptides (in p24, nef, vpr, and gp120).Individual 4 had predicted responses to 47 epitopes contained in thefollowing HIV-1 gene regions: p17, p24, pol, nef, gp120, gp41, vpr, andvif.

Overall, good CTL responses were observed to chosen epitopes (in gag,pol, env, nef, vpr, and vif) in subtype B infected individuals from theUnited States and presumed subtype A/G infected individuals from IvoryCoast.

Mouse Studies

Transgenic HLA mice studies can be carried out to detect immunologicresponses induced by each construct (with and without ubiquitin). Acomparison of the with and without ubiquitin constructs will enablecharacterization of the effects of ubiquitin on epitope processing andimmunogenicity.

One HIV-1 epitope was included in the MCMV-CTL construct (located in themiddle of the construct) which has previously been shown to berecognized by mice expressing MHC class I H-2D. The inclusion of thisepitope allows limited immunogenicity studies with any mouse strain thatexpresses the H-2D allele. In addition, transgenic mice that expresshuman MHC molecules (such as the C57BL/6-TgN(HLA-A2.1) strain) can beused to further look at all of the epitopes in the construct that areA2.1 restricted. By way of example, 1-3 doses of the DNA can be tested,likely 2-5 μg of DNA at a time, applying the DNA interdermally using agene gun.

Mice will be sacrificed and splenocytes will be harvested 7-10 daysafter the last injection. The splenocytes will then be used in Elispotassays to determine if the mice recognize specific peptides containedwithin the construct. Parallel studies can be done with the constructwith and without ubiquitin to demonstrate that the ubiquitin fusionincreases CTL responses (breadth, A2.1 restricted epitopes, andmagnitude).

Primate Studies

Due to the inclusion of the Mamu-A¤01 restricted epitope, monkeys thathave this HLA can be used to study the effects of adding the ubiquitintag, and to characterize the resultant changes in the magnitude of theimmunogenic responses.

B-Cell/T-Helper Cell/Lysosome Constructs

The B-cell (Table 7) and T-helper (Table 8) epitopes were chosen byliterature searches and information and software contained in the LosAlamos HIV Molecular Immunology database. TABLE 7 AA Position B in SEQID epitopes Clade Region Sequence NO: 8 Source Tat A 21-40PCNKCYCKKCCYHCQVCFLN 79-98 Boykins et al. 2001 Tat B 21-40ACTNCYCKKCCFHCQVCFTT  2-21 peptides 21: Tat C 21-40 ACNTCYCKKCSYHCLVCFQT146-165 1839/database Tat D 21-40 PCNKCYCKKCCYHCQVCFIT N/A (SEQ ID NO:46) Tat A/E 21-40 ACSKCYCKKCCWHCQLCFLK N/A (SEQ ID NO: 47) Tat F 21-40PCTKCYCKRCCFHCQWCFIT N/A (SEQ ID NO: 48) Tat A/G 21-40ACSKCYCHICCWHCQLCFLN N/A (SEQ ID NO: 49) Tat A 53-68 KQRRGTPQSNKDHQNP102-117 Tat B 53-68 RQRRRAPQDSQTHQVS 26-41 Tat C 53-68 RQRRSAPPSSEDHQNL170-185 Tat D 53-68 RQRRRPPQGGQAHQDP N/A (SEQ ID NO: 50) Tat A/E 53-68KHRRGTPQSSKDHQNP N/A (SEQ ID NO: 51) Tat A/G 53-68 RRRRGTPQSRQDHQNP N/A(SEQ ID NO: 52) Tat F 53-68 RQRHRTPQSSQIHQDP N/A (SEQ ID NO: 53) gp120HERSYMFSDLENRCI 214-228 2001 Vaccine meeting #295 Menendez et al. gp41 A2F5-4E10 NEQDLLALDKWANLWNWFDIS 122-142 Parker et al. J Virol. gp41 B2F5-4E10 NEQELLELDKWASLWNWFDIT 189-209 2001 75: 10906 gp41 C 2F5-4E10NEKDLLALDKWQNLWSWFDIT 229-249 Non-B subtype gp41 D 2F5-4E10NEKELLELDKWASLWNWFSIT N/A peptide sequences (SEQ ID NO: 54) weredetermined gp41 F 2F5-4E10 NEQELLALDKWASLWNWFDIS N/A using the LosAlamos (SEQ ID NO: 55) HIV sequence gp41 G 2F5-4E10NEQDLLALDKWASLWTWFSIT N/A Database subtype (SEQ ID NO: 56) consensussequence data. gp41 N1 SGIVQQQNNLLRAIEAQQHLLQ N/A Rosny et a. J VirolLTVWGIKQLQARIL 2001 75: 8859-8863 (SEQ ID NO: 57) gp41 C1WMEWDREINNYTSLIHSLIEES 297-330 QNQQEKNEQELL human ECL1 YAAAQWDFGNTMCQLN/A Barassi et al. AIDS CCR5  89-102 (SEQ ID NO: 58) Vaccine 2001 humanECL2 CSSHFPYSQYQFWKNFQTLK N/A abstract #112/ CCR5 178-197 (SEQ ID NO:59) Philadelphia, PA Sep. 5-8, 2001

TABLE 8 AA Position T- in SEQ ID helper Region Sequence NO: 8 Source p24111-132 LQEQIGWMTNNPPIPVGEIYKR 386-407 Wilson et al. J Virol 2001 75:4195 and Cosimi and Rosenberg, Los Alamos HIV Molecular ImmunologyDatabase, 2000 p24 131-152 KRWIILGLNKIVRMYSPTSILD 406-427 Wilson et al.,J. Virol 2001 75: 4195 p24 146-160 SPVSILDIRQGPKEP N/A Cosimi andRosenberg, (SEQ ID NO: 60) Los Alamos HIV p24  1-22PIVQNIQGQMVHQAISPRTLNA 360-381 Molecular Immunology p24 156-170GPKEPFRDYVDRFYK 431-445 Database, 2000 p24 31-52 AFSPEVIPMFSALSEGATPQDL338-359 pol 36-52 EICTEMEKEGKISKIGP 446-462 pol (rt) 303-317FRKYTAFTIPSINNE 467-481 Wilson et al., J. Virol pol (rt) 335-349SPAIFQSSMTKILEP 482-496 2001 75: 4195 pol (rt) 596-610 WEFVNTPPLVKLWYQ497-511 pol (int) 915-929 KTAVQMAVFIHNFKR 512-526 pol (int) 956-970QKQITKIQNFRVYYR 527-541 vpr 66-80 QLLFIHFRIGCRMSR N/A Cosimi andRosenberg, (SEQ ID NO: 61) Los Alamos HIV rev  9-23 DEELIRTVRLIKLLY (SEQID NO: 62) N/A Molecular Immunology rev 41-56 RRRRWRERQRQIHSIS N/ADatabase, 2000 (SEQ ID NO: 63) env 476-490 DMRDNWRSELYKYKV 596-610 env562-576 QQHLLQLTVWGIKQL 611-625 env 667-681 ASLWNWFDITNWLWY 626-640 env682-696 IKIFIMIVGGLIGLR 641-655 env 827-841 HIPRRIRQGLERALL (SEQ ID NO:64) N/A

Construction of a MCMV construct containing these B-cell and T-helperepitopes was carried out essentially similarly to the procedures used togenerate the MCMV-CTL construct. Representative sequences of MCMV-AB/Thconstruct are shown in SEQ ID NOs: 7 and 9; the encoded multivalentantigen polypeptides are shown in SEQ ID NOs: 8 and 10. A tri-amino acidspacer (GPG) was inserted between each of the Ab epitopes and betweenevery 3-5 T-helper epitopes, to provide additional flexibility in themolecule, and to enhance peptide presentation. In addition, the LIMP-Illysosomal targeting sequence was included at the C-terminus in oneconstruct (SEQ ID NOs: 7 and 8), to enhance processing of the epitopes.

Synthetic peptides of the T-helper epitopes have been synthesized, usingstandard peptide synthesis protocols, for use in lymphocyteproliferation assays.

Targeting to Lysosome

Detection of targeting of a MCMV-AB/Th polypeptide to the lysosome canbe accomplished using confocal microscopy. By way of example, HeLa cellscan be transfected with the pVax-1 MCMV constructs (with and without andubiquitin) followed by staining protocols to detect the lysosome(detection of LAMP-1 using a LAMP-1-specific antibody) and the expressedMCMV protein (for example, using polyclonal antibody generated byinjection of the recombinant MCMV protein into mice). A detailedprotocol for the detection of lysosomal targeting is found in Rodriguezet al., J. Virology 75:10421-10430, 2001.

Confirmation of Immunogenicity of Ab and T-Helper Construct(s)

In order to confirm the immunogenicity of the epitopes in a MCMV-AB/Thconstruct, T-helper assays (Lymphocyte proliferation assays) can beperformed using PBMCs from HIV-1 infected individuals, using methodsbasically as described in Wilson et al. J. Virology 75:4195-4207, 2001.

For the antibody epitopes, sera/plasma from infected individuals can beused to test for the presence of antibodies that would react with theprotein encoded by this construct. Additionally, mouse or monkeyimmunization studies with either the DNA construct or purifiedrecombinant MCMV-AB/Th protein (practically any strain of mouse orprimate routinely available) can be preformed to ascertain theproduction of antibodies. Animals would be injected with 1-3 doses ofDNA (2-5 μg DNA for mice and 1-2 mg DNA for rhesus macaques) or purifiedrecombinant protein (20-50 μg for mice or 50-500 μg for monkeys). Priorto the first injection and 1-2 weeks following each injection, bloodwill be drawn and tested for the presence of antibody specific to theMCMV-AB/Th epitopes by ELISA.

Other methods for testing the immunogenicity of Ab and T-helper epitopeswill be known to those of ordinary skill in the art.

Determination of the Optimal Time Frame for Vaccination with theRecombinant Protein as an Immunologic “Boost”

Recombinant protein produced from the MCMV constructs described hereincan be utilized in conjunction with the DNA immunogen(s), or othercurrently available DNA vaccines, as an immunological “boost”.

Following the initial animal injections with the DNA construct(s) immuneresponses will be monitored (for instance, using CTLs, Elispot assays,T-helper/lymphocyte proliferation assays, and/or ELISA assays) todetermine the peak of the immune response for each arm of the immunesystem (T-Cell and B-cell). Based on the observed responses, a series ofboost injections of a MCMV polypeptide can be initiated. By studying theresponses, the time frame to generate the maximum response from memory Tor B-cells can be optimized. Systems for optimizing the boost effectwill be known to those of ordinary skill in the art.

Clinical Trials

Following the production of the vaccine materials, Phase I safety trialscan be performed in populations at risk for HIV. In the United States,target populations would include, for example, gay male cohorts or IVDrug using cohorts. In countries other than the United States, potentialpopulations would include, for example, prenatal cohorts, IV drug usecohorts and prostitutes.

It will be apparent that the precise details of the constructs,compositions, and methods described herein may be varied or modifiedwithout departing from the spirit of the described invention. We claimall such modifications and variations that fall within the scope andspirit of the claims below.

1. An isolated recombinant polyepitope polypeptide comprising aplurality of amino acid segments from one or more HIV-1 proteins,wherein two adjacent amino acid segments are linked by a spacer peptide.2. The isolated recombinant polypeptide of claim 1, wherein the spacerpeptide links multiple groups of amino acid segments.
 3. The isolatedrecombinant polypeptide of claim 1, further comprising a targetingsignal, wherein the targeting signal targets the polypeptide to alysosome or to a proteosome.
 4. The isolated recombinant polypeptide ofclaim 3, wherein the targeting signal comprises a targeting-competentfragment of lysosomal integral membrane protein-II or ubiquitin.
 5. Theisolated recombinant polypeptide of claim 1, further comprising aplurality of amino acid segments from one or more HIV-1 coreceptors. 6.The isolated recombinant polypeptide of claim 5, wherein at least onecoreceptor is CCR5.
 7. The isolated recombinant polypeptide of claim 1,wherein at least one spacer peptide is the tri-amino acidlysine-alanine-alanine, or proline-glycine-proline.
 8. The isolatedrecombinant polypeptide of claim 1, wherein the amino acid segmentscomprise human cytotoxic T-lymphocyte stimulatory epitopes, humanT-helper cell stimulatory epitopes, human B-cell stimulatory epitopes,or combinations of two or more stimulatory epitopes thereof.
 9. Anisolated nucleic acid molecule encoding a polypeptide of claim
 1. 10. Avector comprising a nucleic acid molecule of claim
 9. 11. A host celltransformed with a vector of claim
 10. 12. A composition comprising atleast one polypeptide of claim
 1. 13. The composition of claim 12,further comprising at least one component selected from the groupconsisting of pharmaceutically acceptable carriers, adjuvants, andcombinations of two or more thereof.
 14. A method of eliciting an immuneresponse against an antigenic epitope in a subject, comprisingintroducing into the subject the composition of claim
 12. 15. A methodfor inhibiting or treating HIV-1 in a subject, comprising administeringto the subject the composition of claim
 12. 16. A method for enhancingan immune response in a subject, comprising administering to the subjectthe composition of claim
 12. 17. An isolated recombinant polyepitopepolypeptide comprising an amino acid sequence selected from the groupconsisting of sequences recited in SEQ ID NOs: 2, 4, 5, 6, 8, 10, andcombinations of two or more thereof.
 18. An isolated nucleic acidmolecule encoding a polypeptide of claim
 17. 19. The isolated nucleicacid molecule of claim 18, wherein the nucleic acid molecule comprises asequence selected from the group consisting of sequences recited in SEQID NOs: 1, 3, 7, and
 9. 20. A vector comprising at least one nucleicacid molecule of claim
 19. 21. A host cell transformed with a vector ofclaim
 20. 22. A composition comprising at least one polypeptide of claim17.
 23. The composition of claim 22, further comprising at least onecomponent selected from the group consisting of pharmaceuticallyacceptable carriers, adjuvants, and combinations of two or more thereof.24. A method of eliciting an immune response against an antigenicepitope in a subject, comprising introducing into the subject thecomposition of claim
 22. 25. A method for inhibiting or treating HIV-1in a subject, comprising administering to the subject the composition ofclaim
 22. 26. A method for enhancing an immune response in a subject,comprising administering to the subject the composition of claim
 22. 27.A composition comprising at least one nucleic acid molecule of claim 9.28. A composition comprising at least one nucleic acid molecule of claim18.